Characterization of bacteriophage vB_KleM_KB2 possessing high control ability to pathogenic Klebsiella pneumoniae

Klebsiella pneumoniae is a widespread pathogen of several human diseases. The emergence of multidrug-resistant K. pneumoniae makes the treatment of these diseases a significant challenge. The application of bacteriophages is a potential approach for dealing with the emergence of multidrug-resistant pathogenic bacteria. This study isolates a novel bacteriophage vB_KleM_KB2 that infects the multidrug-resistant clinical isolates of K. pneumoniae. The bacteriophage exhibits a short latent period of 10 min, and can effectively lyse the bacterium within 60 min. Notably, the bacteriophage can completely inhibit the growth of the host bacterium at the initial concentration of 107 CFU/mL with a low multiplicity of infection of 0.001, which proves its high lytic activity. Furthermore, the bacteriophage shows high environmental tolerances, which can facilitate the practical application of the bacteriophage. Analysis of the bacteriophage genome shows that the bacteriophage possesses a novel genome sequence and can represent a new bacteriophage genus. Considering the high lytic activity, short latent period, high stability, and novel genetic background, bacteriophage vB_KleM_KB2 enriches the bacteriophage library and provides a new alternative for controlling the diseases caused by multidrug-resistant pathogenic K. pneumoniae.

Host range of phage vB_KleM_KB2. Among the 15 bacterial strains used for testing the phage host range, only K. pneumoniae strains 2106 and 0915, but not the other strains, can be infected by phage vB_KleM_ KB2. These two sensitive K. pneumoniae strains are β-lactams-resistant and carbapenems-resistant and belong to multidrug-resistant clinical isolates 2 . Although the other two strains of the K. pneumoniae species have also been used for determining the phage host range, they cannot be infected by the phage (Table 1). These results www.nature.com/scientificreports/ indicate that the phage vB_KleM_KB2 maintains high bacterial specificity, which can avoid the influence on normal bacterial flora during practical application in the human body.
one-step growth curve of phage vB_KleM_KB2. The latent time and the burst size of the phage are critical for the practical application of the phage. The analysis of the one-step growth curve shows that the phage vB_KleM_KB2 maintains a latent time of about 10 min. A quick rise of the phage titer is observed between 10 and 60 min after the co-cultivation of the phage with the host bacteria, and a plateaus stage is reached after cocultivating for 60 min with the highest phage concentration up to 10 7 PFU/mL (Fig. 2a). Based on the phage titer at 60 min, the burse size of this phage is calculated to be approximately 63 PFU/cell.
Influence of phage MOI on lytic activity. The lytic activity of the phage is another critical factor that determines the potential for application of the phage. The detection of the bacterial concentration shows that the growth of the K. pneumoniae strains 0915 with the initial concentration of approximately 10 7 CFU/mL is completely inhibited by the phage for at least 360 min, proving the high control ability of the phage to the bacterial strain (Fig. 2b). By treating the K. pneumoniae strains 0915 with phage of different concentrations, even with the MOI as lower as 0.001, the phage still shows a high inhibition ability to the growth of the bacterium, and the growth of the host strain is almost totally inhibited (Fig. 2b). This result indicates that this phage can be used at www.nature.com/scientificreports/ an ultralow MOI, which is attributed to the short latent time and the high burst size of the phage. The ultralow effective MOI can reduce the cost for practical application of the phage.
Stability and environment adaptability of phage vB_KleM_KB2. The other features that influence the practical application of the phage are stability and environmental tolerance. The temperature tolerance analysis shows that even with a temperature as high as 60 °C, the phage vB_KleM_KB2 maintains 90% of the initial viability after being treated for 30 min (Fig. 3a). A sharp decrease in phage viability was observed after being treated at 70 °C, and only 0.05% of the phage remained viable after 30 min of treatment. The phage vB_KleM_KB2 only shows a narrow pH tolerance and can only be stable at pH 7, which is close to the pH of human body fluid (Fig. 3b). The storage stability analysis of the phage reveals that the phage maintains high stability under different temperatures (Fig. 3c). After being kept at 4 °C for 180 days, the phage remains at 15% of its initial viability. After being held at − 20 °C and 28 °C for 30 days, more than 50% of the phage remains viable. The relative low stability at − 20 °C might be attributed to the low resistance of the phage to the freezing and thawing process, which might lead to the inactivation of the phage. In consideration of the high efficiency of the phage in lysing the host bacterial strain at an ultralow MOI, the undemanding requirements on the storage condition would reduce the cost for cold chain transportation of the phage during practical application.
Inhibition effect of phage on the growth of host bacteria under different pH. The pathogenic K.
pneumoniae is found to localize in different environmental conditions. Thus, the ability of the phage to control pathogens under different conditions is needed for practical application. The influence of pH on the inhibition ability of the phage to the host bacteria has been analyzed. The result shows that, at pH 4 and pH 5, the growth of the host bacteria is inhibited by the environmental pH ( Fig. 4). At a pH higher than pH 6, the host bacteria  www.nature.com/scientificreports/ show a normal growth state, and the addition of phage with an MOI of 0.001 can efficiently inhibit the growth of the host bacteria, indicating the phage can be used at a broad pH range from pH 6 to pH 9. Although the phage itself shows low stability by treating with different pH values, the inhibition ability of the phage to the growth of the bacteria is effective at a broad pH, which is because the inhibition on the growth of the bacteria depends on the replication of the phage inside the bacterial cells and is independent from the environmental pH.
Whole-genome analysis. The complete genome of phage vB_KleM_KB2 is a linear double stranded DNA with a genome size of 48,245 bp and G + C content of 48.89% (Fig. 5). The genome of the phage encodes 71 open reading frames (ORFs), of which 44 are transcribed in a forward direction, 27 are transcribed in a reverse direction, and no tRNA genes and no repeats are found in the genome. Besides, no virulence factor is found, guaranteeing the safety of the phage for practical application in the human body. By comparing these genes with the NR database, 67 genes are found to have significantly matched homologous sequences in the database (E-value ≤ 10 -3 ), among which 55 genes show similarity with the genes of Klebsiella phages, including 24 functionally annotated genes, and 4 genes are unique to the phage vB_KleM_KB2. Although the functions of numerous genes in the phage genomes are still unclear, it is believed that genes can be preserved only when they are beneficial to the survival of phages 27 . Among them, 12 ORFs are annotated as phage structural proteins, including gp1, gp3-gp6, gp37, gp38, gp42, gp47, gp49, gp58, and gp68. These genes encode the head protein, neck protein, and tail protein of the phage virion, respectively. A total of 9 ORFs are annotated as DNA metabolism associated proteins, and 3 ORFs are annotated as cell lysis associated proteins. Most functional genes of the phage are modular distribution, and the genes with similar functions cluster together. The genes gp9 and gp70 are annotated as cell lysis protein-encoding genes, which are predicted to encode lysozymes and lytic transglycosylase, respectively. These genes are distributed at termini of the genome and are highly similar to genes of K. pneumoniae phages (> 97% similarity) ( Table S1). The gene gp70 shows high similarity with the genes from Klebsiella phage vB_KpnM_FZ14, 1611E-K2-1, vB_KpnM_KpV52, vB_KpnM_15-38_KLPPOU148, JD001, vB_KpnM_IME346, and MEW1 (Fig. S1). Based on the functions of proteins encoded by genes gp9 and gp70, both work as phage endolysin, which catalyzes the hydrolysis of the peptidoglycan network structure and damages the physical integrity of bacterial cell walls. However, these two endolysins belong to different functional types. The gene gp9 encodes N-acetylmuramidases, and the gp70 gene encodes lytic transglycosylase, which all act on the β-1,4-glycosidic bond between N-acetylteichoic acid and N-acetylglucosamine, but generate different hydrolysates 28 . K. pneumoniae is a gram-negative bacterium with an outer membrane structure www.nature.com/scientificreports/ in the outer layer of its cell wall. The gene product of gp2 is annotated as phospholipase, which can specifically recognize the phosphatidylglycerol and possesses the function to hydrolyze the phospholipid to damage the bacterial cell membrane 29 . The holin-endolysin lysis system exists in almost all dsDNA phages. At the same time, there is no holin-encoding gene in the genome of phage vB_KleM_KB2, indicating that this phage possesses a new holin-encoding gene or harbors a new strategy for lysing bacterial host. There are 9 genes in the phage genome that encode nucleic acid metabolism related proteins, including gp13, gp23-gp24, gp28, gp29, gp33-gp35, and gp40. By searching against the NR database in NCBI, the gene gp23 is predicted to encode ATP-dependent DNA helicase, which plays a catalytic role during the replication of the phage genomic DNA. Gp23 shows the highest similarity (96.54% similarity) with the protein from bacterial strain K. pneumoniae, but exhibits a distant genetic relationship with the protein from the K. pneumoniae phages, including phage vB_KpnM_FZ14, 1611E-K2-1, vB_KpnM_KpV52, vB_KpnM_15-38_KLPPOU148, JD001, vB_KpnM_ IME346, MEW1, and vB_KpnM_KpV79, with similarity less than 56% (Fig. S2A). These results indicate that the gp23 maybe obtain from the host bacterial genome during the infection process. The gene gp24 encodes DNA methyltransferase (MTase), which plays a role in protecting the phage genomes from host encoded restrictionmodification (R-M) systems 30 . Both genes gp33 and gp34 are predicted to encode calcineurin-like phosphoesterase, and the downstream gene gp35 is predicted to encode type I restriction-modification system S subunit. Restriction-modification system is reported to provide bacteria with immunity against phage infection, and the S subunit works in recognizing specific DNA sequences 31 . Together with gp24, these gene products might give the phage resistance to the restriction-modification system of the host bacteria. The gene product of gene gp40 is annotated as HNH endonuclease. What's interesting is that the N-terminal (1-82 aa) of protein Gp40 shows the highest similarity of 81.93% with protein from Klebsiella phage vB_KpnM_IME346, while the C-terminal (79-290 aa) of protein Gp40 shows the highest similarity of 57.73% with that of P. aeruginosa (Fig. S2B). The HNH endonuclease is widely distributed in bacteria, archaea, eukaryotes, viruses, or bacteriophages, which plays a vital role in the genome recombination of the phages 32 . The formation of gene gp40 might be due to the recombination of the gene from P. aeruginosa with a gene from Klebsiella phage.
Comparative genomics analysis of Klebsiella phage vB_KleM_KB2. By searching the NCBI database, 16 phage genomes are found to show more than 50% similarity with the genome of Klebsiella phage vB_ KleM_KB2 (Table S2, Fig. S3). Among these phage genomes, three genes are identified as core genes using the software Coregene 5.0 (Table S3). Except for one gene that is predicted to encode a protein with an unknown function, the other two core genes are predicted to encode baseplate protein and DNA primase, respectively. The phylogenetic tree constructed using these DNA polymerases shows that Klebsiella phage vB_KleM_KB2 and 1611E-K2-1 clustered in the same main evolutionary branch ( Fig. 6a; Table S4). The results of the phylogenetic tree constructed by comparing the whole genome of these 17 phages showed that the phage vB_KleM_ KB2 clustered in the same main evolutionary branch and different sub-branches with Klebsiella phage SBP and Escherichia phage ZCEC13, but it reveals a distant evolutionary relationship (Fig. 6b). Except for Pectobacterium phage PEAT2, which belongs to the Peatvirus genus, the other 15 phages belong to the Jedunavirus genus. As for the whole genome, the genome of phage vB_KleM_KB2 shows the highest similarity with that of phage 1611E-K2-1 (Accession No.: MG197810.1) with 69.4% identical (Fig. 6c). According to the cutoff criteria for genera established by the International Committee on Taxonomy of Viruses (ICTV) Bacterial and Archaeal Viruses Subcommittee, phages are considered to be the same genus if their whole-genome nucleotide sequences exhibit more than 70% identity 33 . Therefore, phage vB_KleM_KB2 could be classified as a new phage genus. The genomes of these phages are analyzed for collinearity and the results show that the genomes of Klebsiella phage vB_KleM_KB2 and vB_KpnM_15-38_KLPPOU148, as well as Pectobacterium phage PEAT2, exhibit entirely consistent linear arrangement (Fig. S4). The results also show that the Klebsiella phages exhibit a general genome rearrangement, indicating the high genome mutation rate of the Klebsiella phages.

Discussion
In this study, a novel phage vB_KleM_KB2 was isolated from the sewage samples. This phage shows high host specificity and only infects two of the tested K. pneumoniae strains, which are both multidrug-resistant clinical isolates, indicating that this phage might be used as an alternative for controlling diseases caused by K. pneumoniae. Compared with the latent periods of the other K. pneumoniae phages, which mainly range from 20 to 70 mins 2,15,16,[34][35][36][37][38][39] , the phage vB_KleM_KB2 exhibits a relatively short latent period of only 10 min. Such a short latent period would benefit the application of the phage by shortening the period for controlling the pathogenic bacteria. This phage also shows an ultralow MOI for controlling the growth of the pathogenic bacteria, and the application of the phage with a low MOI of 0.001 can completely inhibit the growth of the host bacteria. The storage stability is of great importance during the practical application of the phage, as the low storage stability will cause the reduction of control ability of the phage to diseases caused by pathogenic bacteria. It's important that this phage exhibit good environmental tolerance. K. pneumoniae can infect the respiratory tract, intestinal tract, urinary tract, blood, wound, liver, and other parts of the human body 40 , the pH values of which are diverse and range from pH 6.0 to pH 9.0 41,42 . The phage vB_KleM_KB2 shows high lytic activity to the K. pneumoniae strain within the pH range from pH 6.0 to pH 9.0. Considering all these features, the phage vB_KleM_KB2 would be a promising candidate for controlling the diseases caused by K. pneumoniae.
Another feature that benefits the application of this phage is its novel genetic background. Based on the finding that the nucleotide sequence similarity of the whole genome of phage vB_KleM_KB2 is less than 70% with all available phages, the phage vB_KleM_KB2 can be divided into a new phage genus. Compared with the other available K. pneumoniae phages, the phage vB_KleM_KB2 harbors a novel genome sequence with several mutations to facilitate its lytic activity. For example, there are two endolysin encoding genes located at two www.nature.com/scientificreports/ www.nature.com/scientificreports/ termini of the phage genome, which is the reason for the short multiplication period and fast lytic speed of the phage as the extra endolysin would synergistically lyse the cell wall of the host bacterium. Furthermore, gene gp2, which would express at the early stage after phage infection, is annotated to encode the phospholipase. The gene product Gp2 can act on the outer membrane and inner membrane of the K. pneumoniae cell to facilitate the bypass of the endolysin to lyse the bacterial cell wall. This may also be the reason for the rapid and efficient lytic activity of the phage. The novel genome sequence enriches the phage library and provides a candidate for the construction of a "phage cocktail" to avoid the generation of phage resistance. In summary, a novel phage vB_KleM_KB2 that targets the multidrug-resistant clinical isolates of the K. pneumoniae strain has been isolated. The features of the phage vB_KleM_KB2, including high lytic activity, short latent period, high stability, good environmental tolerance, and novel genetic background, make the phage a promising alternative for controlling the disease caused by the multidrug-resistant strains of K. pneumoniae.

Materials and methods
Isolation and preparation of phage. The clinical isolate of multidrug-resistant K. pneumoniae strain 0915 was used as an indicator for isolation of the phage 2 . Sewage samples collected from the Diankun River in Haidian island (Haikou, Hainan, China) were used to isolate the phage that infected K. pneumoniae strain 0915. The sewage samples were centrifuged at 12,000×g for 10 min at 4 °C to remove the solid impurities. Then the supernatants were filtered through a 0.22-μm pore-size membrane filter to remove the bacterial debris. For each sample, the filtered supernatant was added into exponential growth K. pneumoniae strain 0915. After cocultivating at 37 °C for 8 h, the cultures were centrifuged at 8000×g for 30 min, and then filtered through a 0.22μm pore-size membrane filter. The isolation and purification of the phage were carried out using the double-agar overlay method as previously described 43 .

Microscopy observation of phage virions.
To observe the virion morphology, the phage plaques formed on the agar plate were washed off using an SM buffer (10 mM Tris, 100 mM NaCl, and 10 mM MgSO 4 , pH 7.5), and then filtered through a 0.22-μm pore-size membrane filter. The filtered supernatant was collected, and negative staining was carried out with 2% potassium phosphotungstate (pH 7.2) on the copper grid. The morphology of phage particles was observed by transmission electron microscopy (TEM, JEM-1200EX, JEOL, Japan) under 120 kV accelerating voltage.

Determination of host range.
To determine the host range of the phage, bacterial strains of different species, including the species of K. pneumoniae, Acinetobacter baumannii, Escherichia coli, Yersinia pseudotuberculosis, Staphylococcus aureus, and Bacillus pumillus, were used ( Table 1). The determination of the host range was carried out as previously described 2 .
One-step growth curve. A one-step growth curve of the phage was performed to determine the latent period and phage burst size. The phage was mixed with the host bacterium at an MOI of 1.0 for 5 min for absorption. Subsequently, the mixture was centrifuged at 10,000×g for 1 min to remove the non-absorbed phage. Then, the precipitate was resuspended in 50 mL fresh LB broth, and the phage titers in the culture were determined at an interval of 15 min. The one-step growth curve was generated as previously described 44 . Determination of physical stability of the phage. The thermal stability of the phage was determined at 28 °C, 40 °C, 50 °C, 60 °C, and 70 °C. The phage suspension with a concentration of 10 8 PFU/mL was incubated at different temperatures for 30 min and then cooled slowly to room temperature. The phage stored at 28 ℃, nearing room temperature, was used as a control. The phage was incubated in buffer with pH of 1.0, 3.0, 5.0, 7.0, 9.0, and 11.0, for 30 min at 28 °C, and the initial pH value (pH 7.5) of suspension was used as a control. To determine the suitable temperature for storing the phage suspension, the phage suspension was stored at − 20 °C, 4 °C, and 28 °C, for 6 months. The phage titers were determined at 7, 14, 30, 60, 90, 120, 150, and 180 days, after being stored. The phage titer was determined by the double-agar overlay method, and each treatment was performed by three replicates.

Effect of phage concentration on lytic activity.
To determine the effect of different phage concentrations in lysing the host bacterium, the exponential growth K. pneumoniae strain 0915 was used. The phage and the bacterium were mixed with a ratio of 100, 10, 1, 0.1, and 0.01, 0.001, respectively, and left to adsorb for 5 min at 37 °C. After that, the mixture was centrifugated at 10,000×g for 1 min to remove non-absorbed phage. The precipitate was resuspended in 50 mL fresh LB broth and cultivated at 37 °C with moderate shaking. The bacterial culture without adding phage was used as a control. The bacterial concentration was measured every 30 min.

Effect of pH on phage lytic activity.
To determine the influence of environmental pH on the lytic activity of the phage, the pH of the freshly prepared LB broth was adjusted to 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0, respectively, by using the solution of HCl or NaOH. The phage was mixed with the exponential growth K. pneumoniae strain 0915 with a concentration ratio of 0.001. After adsorbing for 5 min at room temperature, the mixture was centrifugated at 10,000×g for 1 min, and the precipitate was resuspended in LB broth of different pH values. The mixture was cultivated at 37 °C with moderate shaking and the optical density (OD 600nm ) of the cultures was measured every 30 min. www.nature.com/scientificreports/ Phage DNA extraction, sequencing, and genomic analysis. As previously reported, the genomic DNA of the isolated phage was extracted with phenol-chloroform after treatment with protease K-sodium dodecyl sulfate (SDS) 45 . The purified phage DNA was sent to sequencing by the Sangon Biotech (Shanghai) Co., Ltd. company with the Illumina HiSeq 2500 sequencer. In total, 22,874,944 reads were obtained and assembled into contigs using the SPAdes-3.5.0 software (Illumina, San Diego, CA, USA). The gaps between the contigs were filled by prime walking. The coding sequences (CDSs) in the genome were predicted by FGENESV0 (Softberry, http:// linux1. softb erry. com) and visualized using SnapGene-V5.2.4.0. Each predicted gene was searched in NCBI non-redundant protein sequences (NR) and CDD databases by using the basic local alignment search tool (BLAST) 46 . The motifs and functional domain compositions of predicted protein were analyzed by Pfam and HHpred database for functional annotation 47,48 . The coding genes of tRNAs were searched by tRNAscan-SE 2.0 (http:// lowel ab. ucsc. edu/ tRNAs can-SE/ index. html) 49 . Tandem Repeat Finder software was used to find the repeats in the genome of phage (https:// tandem. bu. edu/ trf/ trf. basic. submit. html) 50 . Phylogenetic analysis of the functional proteins was performed using MEGA X with the neighbor-joining method and bootstrap analysis (1000 replicates) with the ClustalW alignment 51 . By using the software Coregenes 5.0 (https:// coreg enes. ngrok. io/) 52 , the core genes of phages with more than 50% similarity were obtained (E-value < 10 -5 ). A phylogenetic tree of the whole genome nucleotide sequence was generated using the neighbor-joining method and bootstrap analysis (1000 replicates) in Clustal Omega 1.2.4 (https:// www. ebi. ac. uk/ Tools/ msa/ clust alo/) 53 . Software Mauve 2.3.1 was used for multiple sequence alignment of the phage genomes 54 .

Data availability
The datasets generated and/or analyzed during the current study are available in the NCBI repository www.nature.com/scientificreports/